The present invention discloses a therapeutic vaccine against latent or active tuberculosis infection caused by the tuberculosis complex microorganisms (Mycobacterium tuberculosis, M. bovis, M. africanum). The invention furthermore discloses a multi-phase vaccine that can be administered either prophylactically or therapeutically as well as a diagnostic reagent for the detection of latent stages of tuberculosis. 
Human tuberculosis caused by Mycobacterium tuberculosis (M. tuberculosis) is a severe global health problem, responsible for approx. 3 million deaths annually, according to the WHO. The worldwide incidence of new tuberculosis (TB) cases had been falling during the 1960s and 1970s but during recent decades this trend has markedly changed in part due to the advent of AIDS and the appearance of multidrug resistant strains of M. tuberculosis. 
Organisms of the tuberculosis complex can cause a variety of diseases, but the commonest route of invasion is by inhalation of bacteria. This initiates an infection in the lung, which can ultimately spread to other parts of the body. Normally, this infection is restricted in growth by the immune system, so that the majority of infected individuals show few signs apart from cough and fever, which eventually abates. Approximately 30% of individuals are unable to contain the infection and they will develop primary disease, which in many cases will eventually prove fatal. However, it is believed that even those individuals who apparently control the infection remain infected, probably for the rest of their life. Certainly, individuals who have been healthy for years or even decades can suddenly develop tuberculosis, which has proven to be caused by the same organism they were infected with many years previously. M. tuberculosis and other organisms of the TB complex are unique in that the mycobacteria can evade the immune response and survive for long periods in a refractory non-replicating or slowly-replicating stage. This is referred to as latent TB and is at present a very significant global health problem that is estimated to affect approximately ⅓ of the world's population (Anon., 2001).
The course of a M. tuberculosis infection runs essentially through 3 phases, as illustrated in FIG. 1. During the acute phase, the bacteria proliferate in the organs, until the immune response increases to the point at which it can control the infection, whereupon the bacterial load peaks and starts declining. After this, a latent phase is established where the bacterial load is kept stable at a low level. In this phase M. tuberculosis goes from active multiplication to dormancy, essentially becoming non-replicating and remaining inside the granuloma. In some cases, the infection goes to the reactivation phase, where the dormant bacteria start replicating again. The full nature of the immune response that controls latent infection and the factors that lead to reactivation are largely unknown. However, there is some evidence for a shift in the dominant cell types responsible. While CD4 T cells are essential and sufficient for control of infection during the acute phase, studies suggest that CD8 T cell responses are more important in the latent phase. It is also likely that changes in the antigen-specificity of the response occur, as the bacterium modulates gene expression during its transition from active replication to dormancy.
The only vaccine presently available for clinical use is BCG, a vaccine whose efficacy remains a matter of controversy. Although BCG consistently performs well in animal models of primary infection, it has clearly failed to control the TB epidemic. Consistent with that, BCG vaccination appears to provide protection against pediatric TB (which is due to primary infection), while offering little or no protection against adult disease (which is often reactivation of latent infection acquired in childhood). It has also been shown that vaccination of individuals who are currently sensitized to mycobacteria or latently infected is ineffective. Thus, current vaccination strategies, while effective against primary disease, fail to activate immune responses that efficiently control surviving dormant bacteria.
At this point no vaccine has been developed that confers protection against reactivation whether given as a prophylactic vaccine prior to infection or as a therapeutic vaccine given to already latently infected individuals.
This makes the development of a new and improved vaccine against TB an urgent matter, which has been given a very high priority by the WHO. Many attempts to define protective mycobacterial substances have been made, and different investigators have reported increased resistance after experimental vaccination. However, these efforts have almost exclusively focused on the development of prophylactic vaccines for the prevention of disease (Doherty, 2002), and such vaccines have not been demonstrated to work if given in an immunotherapeutic fashion (J. Turner et al., Infect and Immunity, 2000, pp. 1706-1709).
It has been suggested that the transition of M. tuberculosis from primary infection to latency is accompanied by changes in gene expression (see, for example, Honer zu Bentrup, 2001, which is incorporated herein by reference). In vitro hypoxic culture conditions, which mimic the conditions of low oxygen tension and restricted nutrients found in the granuloma (the location of the latent infection), have been used to analyze changes in gene expression and a number of antigens have been found that are induced or markedly upregulated under these conditions e.g. the 16 kDa antigen α-crystalline (Boon, 2001, Monahan, 2001, Florczyk 2001, Sherman 2001, Manganelli, 2001, all of which are incorporated herein by reference) and Rv0569 as described in Rosenkrands, 2002, and which is described in WO0179274.
As noted in the references cited above, it is already known that some genes are upregulated under conditions that mimic latency. However, these are a limited subset of the total gene expression during latent infection. Moreover, as one skilled in the art will readily appreciate, expression of a gene is not sufficient to make it a good vaccine candidate. The only way to determine if a protein is recognized by the immune system during latent infection with M. tuberculosis is to produce the given protein and test it in an appropriate assay as described herein. Of the more than 200 hundred antigens known to be expressed during primary infection, and tested as vaccines, less than a half dozen have demonstrated significant potential. So far only one antigen has been shown to have any potential as a therapeutic vaccine (Lowrie, 1999). However this vaccine only worked if given as a DNA vaccine, an experimental technique so far not approved for use in humans. Moreover, the technique has proved controversial, with other groups claiming that vaccination using this protocol induces either non-specific protection or even worsens disease (J. Turner et al., Infect and Immunity, 2000, pp. 1706-1709).
What are needed are therapeutic vaccines that treat latent TB infection.